Induction of cell transformation by mutated 16K vacuolar H+-ATPase (ductin) is accompanied by down-regulation of gap junctional intercellular communication and translocation of connexin 43 in NIH3T3 cells

The 16 K subunit of the vacuolar H+-ATPase (ductin) has been suggested to also play a role in gap junction channels. Since mutated 16 K subunits have transforming ability when transfected into NIH3T3 cells and since aberrant gap junctional intercellular communication (GJIC) is a hallmark of cancer cells, we hypothesized that mutated 16 K subunits might transform these cells via alteration of GJIC, When GJIC was measured by the dye-transfer assay, NIH3T3 cells transfected with the mutant 16 K protein genes (deletion of the fourth transmembrane domain or a point mutation at codon 143 from glutamic acid to arginine) showed significantly lower levels of GJIC than those transfected with the vector alone or with the wild-type 16 K subunit gene. GJIC levels of NIH3T3 cells transformed by v-ras and v-src were not significantly decreased, suggesting that low GJIC levels are not necessarily the result of cell transformation pel se. NIH3T3 cells express Cx 43 as a major connexin gene. Although cells transfected with mutated 16 K subunits showed a level of Cx 43 protein expression similar to non-transfectants, their Cx43 protein was localized aberrantly, i.e. intracytoplasmically, These results indicate that mutant 16 K subunits with transforming ability translocate Cx43 proteins, thus inhibiting GJIC of NIH3T3 cells.